Nuclear transfer involves insertion of a nucleus or whole nuclear donor cell (karyoplast) into an enucleated oocyte (cytoplast or recipient cell) followed by fusion of the karyoplast and cytoplast to form a single cell nuclear transfer (NT) embryo. Fusion results in the reprogramming of the donor nucleus by the recipient cytoplasm. Upon suitable activation cleavage division and development may be initiated. Accordingly, an activated single cell NT embryo is a viable embryo, capable of cell division to give a multicellular activated embryo, which is competent to develop in culture to a blastocyst stage.
Activated nuclear transfer embryos may be introduced into the uterus of a synchronised recipient animal, for example, after culture to the blastocyst stage, to give cloned animals.
Nuclear transfer or cloning using somatic cells has been successfully performed in a variety of animals such as cattle (Cibelli et al 1998 Science 280:1256) and sheep (Wilmut et al (1997) Nature 385:810).
A number of standard nuclear transfer techniques employed in species such as cattle and sheep involve electrofusion. When employed in porcine cells, utilizing standard fusion media, such a technique often results in concurrent activation of the recipient cytoplast. Such activation is undesirable at such stage of the procedure. For example, activation induces a large decrease in the levels of maturation promoting factor (MPF) activity in oocytes, high levels of which are generally associated with reprogramming the donor nucleus following fusion. Accordingly, premature activation may interfere with the ability of the cytoplast to reprogramme the donor nucleus leading to decreased developmental competence of the embryo. Premature activation may also trigger other cellular events, such as (pro)nucleus formation, before reprogramming of the donor nucleus was complete.
It is considered that avoiding simultaneous fusion and activation of nuclear transfer embryos may have the advantage of providing the nuclear transfer procedure with flexibility in the type of activation treatment that may subsequently be utilised.
The inventors of the present invention have identified that if electrofusion is conducted using media substantially free of calcium the problem of simultaneous fusion and activation of at least porcine derived NT embryos may be overcome.
Further the inventors of the present invention have surprisingly discovered that in certain cases holding or incubating couplets in media substantially free of calcium for a period prior to electrofusion and NT embryos in a media substantially free of calcium for a period following electrofusion may further help overcome the problem of premature activation of at least porcine derived NT embryos. The inventor's have found this to be particularly applicable where in vitro matured (IVM) oocytes are utilised as cytoplasts.
Accordingly, the invention described herein provides an efficient means of producing at least porcine authentic nuclear transfer embryos.